Research ArticleGastroenterologyInflammation Open Access | 10.1172/jci.insight.171783

Vasantha L. Kolachala,1 Sushma Chowdary Maddipatla,1 Shanta Murthy,1 Yeonjoo Hwang,1 Anne F. Dodd,1 Garima Sharma,1 Sachith Munasinghe,1 Ranjit Singh Pelia,1 Suresh Venkateswaran,1 Murugadas Anbazhagan,1 Tarun Koti,1 Navdeep Jhita,1 Gaurav N. Joshi,1 Chrissy A. Lopez,1 Duke Geem,1 Hong Yin,2 David J. Cutler,3 Peng Qiu,4 Jason D. Matthews,1 and Subra Kugathasan1,3

1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

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1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

Find articles by Qiu, P. in: JCI | PubMed | Google Scholar |

1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

Find articles by Matthews, J. in: JCI | PubMed | Google Scholar

1Division of Pediatric Gastroenterology, Department of Pediatrics & Pediatric Research Institute, Emory University School of Medicine & Children’s Healthcare of Atlanta;

2Department of Pathology, Children’s Healthcare of Atlanta;

3Department of Human Genetics; and

4Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Address correspondence to: Subra Kugathasan, Division of Pediatric Gastroenterology, Emory University School of Medicine & Children’s Healthcare of Atlanta, 1760 Haygood Drive, W-427, Atlanta, Georgia, 30322, USA. Phone: 404.727.1316; Email: skugath@emory.edu.

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

Find articles by Kugathasan, S. in: JCI | PubMed | Google Scholar

Authorship note: VLK, SCM, SM, and YH contributed equally to this work.

Published March 10, 2025 - More info

Published in Volume 10, Issue 5 on March 10, 2025
JCI Insight. 2025;10(5):e171783. https://doi.org/10.1172/jci.insight.171783.
© 2025 Kolachala et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published March 10, 2025 - Version history
Received: April 25, 2023; Accepted: January 16, 2025 View PDF Abstract

Crohn’s disease (CD) involves a complex intestinal microenvironment driven by chronic inflammation. While single-cell RNA sequencing has provided valuable insights into this biology, the spatial context is lost during single-cell preparation of mucosal biopsies. To deepen our understanding of the distinct inflammatory signatures of CD and overcome the limitations of single-cell RNA sequencing, we combined spatial transcriptomics of frozen CD surgical tissue sections with single-cell transcriptomics of ileal CD mucosa. Coexpressed genes and cell-cell communication from single-cell analyses and factorized genes from spatial transcriptomics revealed overlapping pathways affected in inflamed CD, like antigen presentation, phagosome activity, cell adhesion, and extracellular matrix. Within the pathways, early epithelial cells showed evidence of significant changes in gene expression and subtype composition, while spatial mapping revealed the location of the events, particularly antigen presentation from epithelial cells in the base of the crypt. Furthermore, we identified early epithelial cells as a potential mediator of the MHC class II pathway during inflammation, which we validated by spatial transcriptomics cell subtype deconvolution. Therefore, the inflammation from CD appears to change the types of interactions detectable between epithelial cells with immune and mesenchymal cells, likely promoting the conditions for more macrophage infiltration into these inflammatory microdomains.

Introduction

There is an unmet need to better define the molecular and cellular landscape of Crohn’s disease (CD), an inflammatory disease of the gastrointestinal tract that currently affects nearly 3 million individuals in North America (1–4). The heterogeneous nature of CD is related to the intricate and complex microenvironments of the intestine, where the immune and stromal compartments are separated from luminal contents and microbiome by an epithelial barrier. Factors including genetics, diet, smoking, and environmental conditions appear to contribute to the epithelium’s inability to maintain this barrier in CD, promoting a microenvironment of chronic inflammation with ongoing mucosal injury (5). Cellular signaling via receptor-ligand (RL) interactions between different cell types in the mucosa has been well established, with anti-TNF therapy being the most successful treatment to date that targets these processes. However, anti-TNF response remains transient in many patients with CD, with most patients eventually developing antibody resistance. Consequently, there is a need to better understand the mechanisms underlying persistent inflammation in CD.

Substantial gains in characterizing the cellular basis of inflammatory bowel disease (IBD) have been achieved using single-cell technologies (6–10). Despite this, the spatial context of cells in the mucosa is lost during single-cell preparation, making it difficult to localize the activity and interactions of cells. Thus, a more accurate view of the architectural, cellular, and molecular changes taking place in the inflamed ileal mucosa of CD can be made using spatial transcriptomics (ST) on resected surgical tissues in conjunction with single-cell RNA sequencing.

Here, we combine single-cell RNA-sequencing and ST approaches to enhance our view of cellular changes taking place in the ileal mucosa during active CD. Using cell-cell communication (11), coexpressed gene modules (12), and spatial factors (13, 14), we substantiated pathways represented during active CD and mapped their activity onto ileal resected tissues. Through our integration of single-cell and ST approaches, we uncover cell type activity and gene expression patterns varying between inflamed and noninflamed CD.

Results

Single-cell profiling of ileal mucosal cells derived from patients with Crohn’s. To gain insight into the inflammation-associated cellular dynamics during CD, we performed single-cell RNA sequencing on ileal biopsies obtained from patients with CD, with n = 8 inflamed CD and n = 7 noninflamed CD, at the time of colonoscopy (Figure 1A and Table 1). After single-cell data preprocessing and quality control (Supplemental Figure 1, A–D; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.171783DS1), the obtained 52,396 cells were clustered into 3 intestinal compartments — epithelial, immune, and stromal — that were annotated by transcriptional signature differences. Fine clustering and manual annotation based on canonical marker genes’ expression further identified 28 cell types (Supplemental Table 1). Of these cell types, 16 were epithelial, 11 were immune, and 1 was a stromal subpopulation (Figure 1, B and C). Next, we delineated relationships between cell types using trajectory analysis with partition-based graph abstraction (PAGA). This approach supported our manual annotations by quantifying the connection strengths between annotated cell types within epithelial and immune subpopulations (Figure 1D).

Figure 1

Experimental setup and single-cell transcriptomic analysis. (A) Schematic of ileal biopsy collection and single-cell and spatial transcriptomic analysis. (B) Uniform manifold approximation and projection (UMAP) of integrated and annotated single-cell transcriptomic data (n = 15), with 28 annotated cell types. (C) Heatmap showing the expression of cell type–specific marker genes. (D) Inferred trajectory of epithelial and immune differentiation.

Table 1

CD patient metadata for ileal biopsies processed for single-cell transcriptomics

Epithelial and B cell subtypes exhibit proportional and transcriptional changes associated with ileal mucosal inflammation during CD. Using a list of published inflammation-associated genes, we evaluated inflammation scores across cells in inflamed and noninflamed CD (Figure 2A and Supplemental Figure 1E). We did not find global differences in this score between inflamed and noninflamed CD mucosa (Figure 2B). However, we detected a significant increase in inflammatory scores in the stem cells, transit-amplifying cells, goblet cells, and enterocytes in the epithelium, along with B cells in the immune compartment in inflamed CD. When examining cellular proportions between the noninflamed and inflamed mucosa, we observed a marked decrease in absorptive epithelial lineage and a substantial increase in immune populations (Figure 2C). Specifically, the relative proportions of absorptive epithelial cells such as immature enterocytes and enterocytes, mature enterocytes, BEST4+ cells, and enteroendocrine cells were significantly reduced, while the abundance of plasma cells, a B cell subtype, increased during inflammation (Figure 2D).

Figure 2

Cellular and transcriptional changes associated with mucosal inflammation in CD. (A) UMAP overlay colored by inflammation status. (B) Inflammation score density for inflamed and noninflamed separately in overall mucosa (leftmost plot) and box plots of selected cell types showing significant difference in inflammation score between the 2 groups (Wilcoxon’s rank sum test, P value < 0.05). (C) Bar plot showing cell lineage proportions between inflamed and noninflamed groups in overall mucosa. (D) Box plots of the selected cell types with significance (Student’s t test, P value < 0.05).Box plots show the interquartile range, median (line), and minimum and maximum (whiskers).

Antigen presentation and cell adhesion molecules are promoted in early epithelial cells and lymphocyte subtypes during mucosal inflammation. To further understand gene expression patterns during active inflammation in CD, we used gene modules to investigate coexpression of (DEGs) within each cell subtype. We first identified 11,480 unique significant DEGs within the cell types from the inflamed ileal mucosa, with Paneth cells and mature enterocytes exhibiting the largest changes in gene expression (Figure 3A and Supplemental Table 2). DEGs were then clustered to determine those that were coexpressed and could be defined as modules (Supplemental Table 3) (12). Gene set enrichment analysis was conducted on these modules to determine pathways associated with inflammation. Frequencies of each enriched pathway were calculated for both epithelial and immune cell types. The frequently enriched pathways within the epithelial cells included antigen processing and presentation, phagosome, intestinal immune network for IgA production, and cell adhesion molecules (CAMs). In modules from immune cell types, the frequently enriched pathways included VEGF signaling, phagosome, and NOD-like receptor signaling, though the frequencies of these pathways were lower than those in the epithelial cell modules. Of note, antigen processing and presentation, phagosome, and CAM pathways were also enriched for immune cells (Figure 3B and Supplemental Figure 2, A and B).

Figure 3

Antigen presentation and cell adhesion molecules are promoted during mucosal inflammation. (A) Bar plot of the number of significantly (adj. P value < 0.05) differentially expressed genes (DEGs) in inflamed (purple) versus noninflamed (orange) cells per cell type using the MAST algorithm. (B) Bar plots of the frequency of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways significantly (P value < 0.05) enriched in epithelial (pink) and immune (blue) cell type modules of the DEGs. (C) Heatmaps of key epithelial module scores across the cell types in inflamed and noninflamed groups. Dots represent enrichment. (D) Heatmaps of key immune module scores across the cell types in inflamed and noninflamed groups. Dots represent enrichment.

The most frequently enriched pathways were further explored by calculating module scores based on the genes contributing to each epithelial module pathway (Figure 3C). Module scores for CAMs and intestinal immune network for IgA production were increased in inflamed stem cells, TA cells, and immature enterocytes. In contrast, focal adhesion and ECM-receptor modules were markedly enriched in noninflamed stem and TA cells. The same approach was taken to examine the frequently enriched module pathways from immune cell types (Figure 3D). The NOD-like receptor signaling pathway module was enriched in inflamed T cell populations, and the phagosome was enriched in inflamed stem, TA, and immature enterocyte cells. In contrast, the VEGF signaling pathway was enriched in noninflamed stem, TA, immature enterocyte, and goblet cell populations. Together, focal adhesion pathway, ECM pathway, and VEGF pathway were less pronounced during active inflammation. These signatures may indicate pathways involved in downregulating inflammation or could be signs of mucosal healing in the ileum within individuals with CD.

Several HLA genes were detected across module pathways. The DEGs contributing to these pathways were assessed per cell type (Supplemental Figure 2C). Stem and TA cells had significantly increased expression of HLA-related module genes (HLA-DPA1, HLA-DPB1, and HLA-DQB1) during active inflammation. Additionally, BIRC3 and JUN from the focal adhesion module were significantly higher in T cells and B cells during inflammation, respectively. Taken together, these signatures point to pathways, particularly in the epithelial cells in the base of the crypts, that are undergoing changes during inflammation of the ileal mucosa.

Global cell communication analysis reveals distinct differences in signaling networks across cell populations during inflammation in CD. Given the changes in antigen processing and presentation and cell adhesion activity during inflammation, we next investigated the intercellular communication differences between inflamed and noninflamed CD samples. Using CellChat, we analyzed interactions among 28 cell populations in inflamed and noninflamed groups (Supplemental Table 4), as well as in individual CD patient samples (Supplemental Figure 3), to better understand the patient heterogeneity. We examined the total number of incoming and outgoing interactions and cellular compartments most perturbed by inflammation. Our initial analysis showed a similar total number of interactions in both groups (more than 15,000 RL pairs) (Figure 4A). However, at the cellular-compartment (epithelium, immune, and stromal) level, the inflamed mucosa exhibited a loss of 560 predicted epithelial interactions, alongside an increase of 92 immune and 9 stromal interactions, compared with noninflamed. Notably, the interactions of stromal cells to both immune and epithelium, followed by epithelium to immune, were predicted to be stronger within the inflamed CD mucosa (Figure 4B).

Figure 4

Altered putative cell signaling networks in inflamed compared with noninflamed CD. (A) Bar plot of total number of possible RL interactions. (B) Number of RL interactions between the 3 major compartments. (C) Heatmap illustrating the selected signaling patterns in inflamed and noninflamed CD. (D) Chord diagrams depicting the MHC class II (MHC-II) signaling network (left 2 plots) and dot plot showing RL signaling toward naive T cells uniquely in inflamed (right). Edge color in the chord plot represents the signaling source, and segments with arrows are the signaling targets.

We then examined pathway patterns by calculating each cell type’s incoming and outgoing signal strength. This analysis revealed several pathways that were unique to inflamed ileal CD, including interleukin-1 (IL1), vascular cell adhesion protein (VCAM), and THY1 membrane glycoprotein. In contrast, pathways like protein S, CD226, and growth arrest-specific protein networks were uniquely enriched in noninflamed ileal mucosa. Notably, pathways linked to epithelial proliferation and differentiation, such as BMP, NOTCH, and WNT, were enriched in noninflamed CD, while ncWNT was upregulated in inflamed CD. We also observed increased signaling of key inflammatory pathways, including APP and MHC class II (MHC-II), in inflamed CD. Additionally, pathways involved in ECM, including AGRN, FN1, CHAD, and HSPG, were upregulated in inflamed CD (Figure 4C and Supplemental Figure 4).

Next, we assessed the flow of information among cell populations involved in the MHC-II (antigen presentation) pathway based on communication probability. In inflamed CD, the chord diagram shows naive T cells as the signaling target and early epithelial cells as the signaling source, a pattern absent in noninflamed tissue. Additionally, the RL pairs mediating this signaling toward naive T cells were elevated across several cell types in inflamed CD (Figure 4D).

Inflamed Crohn’s ileal mucosa indicates dynamic alterations in intercellular signaling. We then investigated the signaling mechanisms that were potentially altered during inflammation. We identified dysregulated RL pairs involved in epithelial differentiation pathways during inflammation. We detected changes in BMP and ncWNT signaling, pathways crucial for maintaining intestinal crypts. We also found that the source of BMP2 and BMP4 ligands shifted from stromal cells and epithelial subtypes in noninflamed mucosa to primarily epithelial subtypes in the inflamed. Additionally, the WNT5A ligand in the noncanonical WNT pathway, expressed by Paneth and TA cells in noninflamed mucosa, was primarily expressed by enterocyte subtypes and stromal cells during inflammation (Figure 5A).

Figure 5

Dynamic alterations in signaling pathways in the inflamed Crohn’s ileal mucosa. (A) Inferred BMP and ncWNT RL communications between stromal and epithelial cell types. BMP, bone morphogenetic protein; nc, noncanonical. (B) Inferred CCL and CXCL RL pairs across multiple cell types.

Chemokines essential for wound healing were predicted to be altered during inflammation. In the CCL network, macrophages and inflammatory macrophages emerged as signaling targets through the CCR1 receptor in the inflamed mucosa. Likewise, in the CXCL pathway, B cell subpopulations, naive T cells, Tregs, and macrophages became signaling targets in the inflamed mucosa. This contrasts with the noninflamed mucosa, where macrophages and CD8+ T cells were the cellular targets during inflammation (Figure 5B). Together, our findings predict dynamic alterations in signaling strength and RL communication architecture during inflammation.

Non-negative matrix factorization demonstrates spatial compartmentalization and localizes active pathways represented in CD. Next, we conducted ST on 16 surgically resected ileal tissues from 4 inflamed and 1 noninflamed CD patients (Table 2 and Supplemental Figure 5) that were all processed using the 10x Genomics Visium platform. We first explored the basic transcriptomic patterns underlying each tissue using 3 factors generated by non-negative matrix factorization (NNMF) (13). The 3 factors distinguished the epithelium, lamina propria/mixed, and smooth muscle compartments (Figure 6A and Supplemental Figure 6) (13). Factors were compared across the same patient’s tissue sections obtained serially. Annotated factors were highly correlated and offered evidence of similar transcriptional activity being retained in tissues from the same patient (Figure 6B and Supplemental Table 5). This correspondence suggests that the ST data provide robust representation of patient mucosal biology. The labeled compartments were also verified by antibody staining using immunofluorescence and transcriptomic expression of canonical cell markers (Figure 6C). To uncover more distinctive, regionalized activity that was sufficiently represented across the slides, we conducted pathway enrichment analysis on 10 detailed factors per slide and assessed those that were most frequently enriched (Figure 6D). Frequently enriched pathways included antigen processing and presentation, phagosome, ECM-receptor interactions, CAMs, and focal adhesions that were also consistently observed when examining pathways per patient (Figure 6E).

Figure 6

Non-negative matrix factorization delineates compartmentalization and regionalization of disease-implicated pathways. (A) Mapping of non-negative matrix factorization (NNMF) (n = 3 factors) genes onto representative spatial tissues ST15 and ST16 from patient 5. LP, lamina propria. (B) Correlation of factors across spatial tissue sections from patient 5. (C) Immunofluorescence (left) and RNA expression (right) of markers corresponding to compartments. a-SMA, α–smooth muscle actin. (D) Top pathways enriched across all patients using NNMF (n = 10 factors per slide). (E) Dot plot representing enriched pathways corresponding to factors per patient tissue.

Tabl